Cadherin-11 in renal cell carcinoma bone metastasis

Abstract

Bone is one of the common sites of metastases from renal cell carcinoma (RCC), however the mechanism by which RCC preferentially metastasize to bone is poorly understood. Homing/retention of RCC cells to bone and subsequent proliferation are necessary steps for RCC cells to colonize bone. To explore possible mechanisms by which these processes occur, we used an in vivo metastasis model in which 786-O RCC cells were injected into SCID mice intracardially, and organotropic cell lines from bone, liver, and lymph node were selected. The expression of molecules affecting cell adhesion, angiogenesis, and osteolysis were then examined in these selected cells. Cadherin-11, a mesenchymal cadherin mainly expressed in osteoblasts, was significantly increased on the cell surface in bone metastasis-derived 786-O cells (Bo-786-O) compared to parental, liver, or lymph node-derived cells. In contrast, the homing receptor CXCR4 was equivalently expressed in cells derived from all organs. No significant difference was observed in the expression of angiogenic factors, including HIF-1α, VEGF, angiopoeitin-1, Tie2, c-MET, and osteolytic factors, including PTHrP, IL-6 and RANKL. While the parental and Bo-786-O cells have similar proliferation rates, Bo-786-O cells showed an increase in migration compared to the parental 786-O cells. Knockdown of Cadherin-11 using shRNA reduced the rate of migration in Bo-786-O cells, suggesting that Cadherin-11 contributes to the increased migration observed in bone-derived cells. Immunohistochemical analysis of cadherin-11 expression in a human renal carcinoma tissue array showed that the number of human specimens with positive cadherin-11 activity was significantly higher in tumors that metastasized to bone than that in primary tumors. Together, these results suggest that Cadherin-11 may play a role in RCC bone metastasis.

Figure 1. Generation of organ-tropic 786-O RCC cells.

(A) Parental 786-O RCC cells were labeled with luciferase and GFP. (B) Images of bioluminescence of mice at indicated time point after intracardiac injection with parental 786-O cells. (C) 786-O cells derived from liver, lymph node and bone, were GFP-positive.

Figure 2. Expression of Cad11 in 786-O cell lines derived from metastases to various organs.

(A) Quantitative PCR for the message levels of Cad11 in the four 786-O cell lines. (B) Western blotting for the protein levels of Cad11 in four 786-O cell lines. Upper panel: A representative image of Western blot. Lower panel: Quantification of band density using Image J software. Data were expressed as folds of parental 786-O cells and the values were the Mean ± S.E. n = 5. *: p<0.05; **: p<0.01 as compared to parental 786-O cells. (C) FACS for surface expression of Cad11 in the four 786-O cell lines. Data were expressed as percentage of gated cells. (D) Immunofluorescence staining of cells with anti-Cad11 antibody. P, Parental 786-O; Liv, Liv-786-O; LN, LN-786-O, and Bo, Bo-786-O RCC cells.

Figure 3. Expression of CXCR4 in 786-O cell lines.

(A) Quantitative PCR for the message levels of CXCR4 in the four 786-O cell lines. *: p<0.05; **: p<0.01 as compared to parental 786-O cells. (B) Western blotting for the protein levels of CXCR4. Left panel: A representative image of Western blot. Right panel: The quantification of band density. Data were expressed as folds ± S.E. of parental 786-O cells. n = 6. (C) The number of cells with CXCR4 expression in cell surface was determined by FACS. Data were expressed as percentage of gated cells (right upper panel) and as the median of fluorescence intensity (right lower panel).

Figure 4. Message levels of angiogenic and osteolytic factors in 786-O RCC cell lines.

Quantitative PCR for the message levels of angiogenic factors HIF-1α (A), VEGF (B), Ang1 (C), Tie-2 (D) and c-Met (E), and osteolytic factors PTHrP (F) and IL-6 (G) in the four 786-O cell lines. Data were expressed as folds of parental 786-O cells and the values were the Mean ± S.E. *: p<0.05; **: p<0.01 as compared to parental 786-O cells.

Figure 5. Effect of Cad11 on cell proliferation and migration of Bo-786-O cells.

(A) Proliferation and migration of parental and bone-derived 786-O cells. Left: Western blot of Cad11 protein. Middle: Cell proliferation. Right: Cell migration. A representative image of cell migration and the quantification of cells that migrated to the other side of migration inserts were shown. Values for migration were expressed as the average of migrated cells per microscope field (X100). (B) Effect of Cad11 knockdown on the proliferation and migration of Bo/shCont and Bo/shCad11 cells.

Figure 6. Immunohistochemical staining of human RCC specimens with anti-Cad11 antibody.

(A) Representative image of primary RCC; (B) Representative image of bone metastatic RCC.